Abstract

Background/Objectives: Adoptive transfer of allogeneic natural killer (NK) cells represents a promising off-the-shelf immunotherapy for cancer, offering advantages in safety and availability over autologous T cell therapies. However, generating therapeutically sufficient NK cell numbers remains challenging due to their low frequency in blood sources. Engineered feeder cell co-cultures have enabled substantial expansions of NK cells to clinically relevant doses. Methods: We evaluated the plasma cell leukemia-derived ARH-77 cell line as a feeder for ex vivo NK cell expansion from healthy donor peripheral blood mononuclear cells (PBMCs). Unmodified ARH-77 was compared to K562, followed by engineering both lines to co-express B7-H6 (NKp30 ligand), CD137L (4-1BBL), IL-15, and IL-15Rα via sequential lentiviral transduction. PBMCs were co-cultured with irradiated feeders in cytokine-supplemented (IL-2, IL-21, and later IL-15) RPMI-1640 or DMEM/F-12 medium for up to 28 days. Expansion (fold change in CD3−CD56+ cells), purity, surface receptor expression, and cytotoxicity (against K562 targets) were quantified. Results: Unmodified ARH-77 supported significantly greater NK cell expansion than K562 (model-estimated 681-fold vs. 155-fold at week 4 in RPMI; p = 0.0018), with higher purity but comparable cytotoxicity and receptor profiles. Engineered ARH-77 cells achieved robust expansion in RPMI, comparable to that of engineered K562 cells. In optimized DMEM/F-12 medium, engineered ARH-77 drove superior expansion (up to model-estimated 101,241-fold; 95% CI 46,771–219,146 at week 4), significantly outperforming engineered K562 (4.4-fold greater; 95% CI 1.01 to 18.54; p = 0.0479) while maintaining high purity and equivalent cytotoxicity. Substantial inter-donor variability influenced expansion magnitude, though relative feeder performance remained consistent across donors. Conclusions: Genetically modified ARH-77 feeder cells provide a potent platform for large-scale ex vivo expansion of functional NK cells.

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